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Funguy182
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Registered: 09/04/17
Posts: 407
Last seen: 5 years, 11 days
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Gentamicin question
#25008093 - 02/20/18 01:14 PM (6 years, 3 days ago) |
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Just wondering if anybody has used Gentamicin in their liquid culture Media or in there agar media? If so what is the recommended dose for say 600ml of water for the lc media and 200 ml for agar media. Also bought some peptone was wondering about dosage amounts of that for the same. Thanks
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ChemBioWiz
Laboratory Explorer

Registered: 01/27/18
Posts: 39
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Re: Gentamicin question [Re: Funguy182]
#25010102 - 02/21/18 08:52 AM (6 years, 3 days ago) |
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My first question would be what your conditions/motivations are to make you feel like you want an antibiotic? Generally speaking, most edibles/medicinals tend to colonize media relatively quickly as long as it's prepared carefully. What's more, Joe Everyman probably shouldn't be contributing to antibiotic resistance in the environment; a lot of folks who use antibiotics at home dump their used media down the drain or into the common waste stream, which is no bueno. I would recommend refining procedure, or adjusting media nutrient concentration and pH before jumping into antibiotic work. But hey, if there's a need, you're going to dispose of it properly, and that's where you want to take this train, who am I to stop you?
I think I've read some threads where folks were talking about scooping a small spatula-tip of the solid form into a large volume of liquid media and going at things like that. Now that's a bit careless if you ask me, but my tolerance for error is a bit lower because I'm a chemist by profession. Just one man's opinion. Realistically, the precision/accurate measurement equipment to do the job correctly is expensive for the average home-grower so folks are making due with what they've got, and there's some wiggle-room in the final concentration. I'll give you a take on this from my perspective:
When working with antibiotics, a person really should have some decent measurement equipment (e.g. 0.0001 g scale) to parse out the powdered-form and quality volumetric equipment to help dilute solution-forms of the antibiotic effectively (e.g. 5 mL, 10 mL, 1 L graduated cylinders; 1 mL plastic transfer pipettes or a 100-1000 µL pipette). There's always the option to have a lab supply company compose a custom liquid concentration, but that's well beyond most people's price-range. If someone can afford that, they're better off investing in quality equipment.
If there is a research university or well-stocked college lab close by, try asking if they will help mix up a stock solution from the powdered sulfate and dilute a liquid stock down to a more manageable concentration. Mailing it back and forth would be difficult and expensive because the stuff has to be kept cold (≈-2 to -20°C). Who knows, they might even let you use their autoclave, which would save a lot of time and effort.
The nitty-gritty: Merck's posted recommendation for Gentamicin sulfate has the user creating a 50 mg/mL stock and diluting down; pretty standard stuff, but to be clear: that's too high a concentration to use by itself. ResearchGate lists the working concentration at 1.0 µg/mL, AddGene and ProtocolsOnline list it at 10 µg/mL, and Thermo-Fuckoverthecustomer puts it between 0.5-50 µg/mL. Without digging into the literature, I'd guess the minimal bactericidal concentration (MBC) is probably somewhere at the low end of Thermo's range. That should provide a good conceptual bubble for the stuff. If you're as dirty as Alexander Fleming was, 25 µg/mL is probably a safe bet.
If someone is working with the solid, they're going to want to dilute it into a stock solution first, which is easier to work with than the powder directly. That's going to be difficult without an accurate scale handy.
There are liquid stock solutions out there for sale; they tend to be more expensive and don't have as long a shelf-life, but they're easier to manage accurately using a cheap, 1 mL plastic transfer pipette and 5 mL graduated cylinder. The graduations are usually 0.2 mL and 0.1 mL, respectively. Once the stock solution is in hand, a person can use the dilution equation (C1V1=C2V2) to get the concentration they need for their final volume of liquid media/agar.
Quote:
C1 is the starting concentration of stock antibiotic solution.
V1 is the unknown 'x' value (how much of that stock we want to add to the media).
C2 is the ending antibiotic concentration in the media.
V2 is the final media volume (you said 600 or 200 mL).
Using a standard 50 mg/mL antibiotic stock solution with a final concentration of 25 µg/mL and adjusting so the units are the same, the equation will look like this:
Quote:
(50 mg)(x) = (0.025 mg)(600 mL)
x = [ (0.025)(600) ] / 50
x = 0.3 mL
So that means 0.3 mL of the 50 mg/mL stock solution would need to be added to the 600 mL of media to get a working concentration of 25 µg/mL. For the agar, 600 mL is replaced with "200 mL." Normally the final volume includes the volume of added antibiotic (e.g. a total volume of 600 mL), but the addition is so small that the average home-grower isn't going to be able to accurately measure that small a volume. They don't have to worry about it for their purposes anyway.
Just one final bit of information: Gentamicin sulfate is only stable in solution under 37°C incubation conditions for around 120 hours (5 days). That's the manufacturer's listed limit. Most home-growers are probably working at room temperature (≈25°C) so that will extend a bit longer. I wouldn't push it much past 10 days. I recall a study floating around out there where the researchers went to 15 days reliably, but you'll have to double-check that one. Also, if agar plates are being stored at 0°C, they will keep for quite a while before degrading.
I hope that helps. Drop me a line if you need me to go into more detail. Correct me if I messed anything up, it won't hurt my feelings.
-------------------- Mycology is a cool community of people. Try not to add any bad vibes to the neighborhood. Finding mushrooms in the woods and growing them at home brings me a lot of joy.
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Funguy182
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Registered: 09/04/17
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Last seen: 5 years, 11 days
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Thank you for your insight on this matter. I really am not having trouble with contamination but I read it is used as a preventative measure. I also read if mixed with agar solution it keeps contams back long enough for the intended fungus to thrive. This is mostly just curiosity on my part. I'm not a big fan of antibiotics myself unless needed. My real question would be about peptone. I just ordered some today and I read stamets book and he uses it very liberally in his agar mixtures. I'm not math genius but his formulas are intended for larger scale production and he gives a formula to break it down but I am fucking clueless on how to figure that out. My question is how much peptone do I add for 200 ml of agar solution and how much for 600 ml lc solution. I have read peptone increases strength and growing speed. Wich is always good. Especially for the slow growers like Lentinula Edodes. My pH is on Target and my process is as clean and steril as it gets for being a home grower. allot of bleach solution and 70% iso alc. I appreciate your response in such detail and insight. I own my own growing business and I'm still learning new things. Anything that helps is always welcomed.
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Humble Newcomer
Diddler de niños



Registered: 03/12/17
Posts: 1,483
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Re: Gentamicin question [Re: Funguy182]
#25011948 - 02/21/18 09:53 PM (6 years, 2 days ago) |
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I'm just a fly on the wall here, but intrigued by y'all's conversation hoping to pop in and quietly learn, funguy, may I ask why you're intending on a peptone recipe?
Im for the first time looking to modify my agar recipe in certain circumstances (color) and I'm intrigued in grain water or cow manure water agars perhaps, something dark yet free and abundant for me to obtain.
But I've been running super simple potato flake pda and having no issues with multiple species, I have two shiitake now thanks to a friend, a log culture and 3782, and the latter just tears through my cheap pda almost at oyster speed.
Im curious how much hassle it is to make these wild recipes I see, and what people's reasoning is because I had expected way more problems and haven't run into any yet. Thanks
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Funguy182
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Registered: 09/04/17
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Last seen: 5 years, 11 days
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I'm wanting peptone bc it's supposed to show explosive growth on agar. Since I run a business it is convenient. My agar works great growth explodes already but always looking for better. If you are looking for dark but clear agar recipe I heard t-gel agar works good. I personally use dextrose and malt extract plus peptone. I do 200 ml batches. I prefer stiff agar so I use a little more than required. I think it prolongs the life of the agar. I do 2/8 tsp of malt extract 4 tsp agar 1/2 tsp dextrose and 1/2 tsp peptone. So far it's showing great results. I use one drop of blue fc it makes for a super light blue perfect for seeing any undesirables.
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ChemBioWiz
Laboratory Explorer

Registered: 01/27/18
Posts: 39
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Re: Gentamicin question [Re: Funguy182]
#25051214 - 03/09/18 06:45 AM (5 years, 11 months ago) |
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Been tied up in the lab lately, but finally had a moment to respond.
Quote:
Funguy182 said:
...I also read if mixed with agar solution it keeps contams back long enough for the intended fungus to thrive. This is mostly just curiosity on my part. I'm not a big fan of antibiotics myself unless needed....
Fungi are amazing organisms. Chances are they're going to "thrive" without antibiotics if your sterile technique is on point. Still, you should expect to experience some failure even after doing everything meticulously/correctly; sometimes it just comes down to organismic factors throwing us a curveball. We all want to avoid getting it wrong if we can help it, but that's the best way to learn. I would recommend playing around with your concentrations and pHs, and intentionally contaminating a few things to see how the cultures you're working with respond to changes in those variables.
Quote:
Funguy182 said:
...My question is how much peptone do I add for 200 mL of agar solution and how much for 600 mL LC solution.
The dilution equation works well for transfers of media in solution, but what if you're starting with a solid? It's not that complex, but if you don't engage with maths like this that often it can seem somewhat problematic. The solution involves setting up a ratio and cross-multiplication.
Let's say the Agar recipe in question involves adding 1 g of solute (the peptone) to a final volume of 1000 mL of solution. That means there is a concentration of 1 g / 1000 mL. You want to know how much solute you need to recreate that concentration using only 200 mL of solution.
Quote:
You have this recipe: 1 g / 1000 mL
You want this recipe: x g / 200 mL
1. Set them equal to each other 2. Cross-multiply 3. Isolate x 4. Divide and solve
1. 1 g / 1000 mL = x g / 200 mL ⇒
2. (1000 mL)(x g) = 200 mL ⇒
3. x g = 200 mL / 1000 mL ⇒
4. x = 0.2 g
We can change the numbers out for the starting and final concentrations, depending on what sort of recipe we're working with.
An important thing to remember here is that those mL values are for the final volumes. Adding solute to a pre-poured solvent will increase the volume slightly, throwing off the concentration that was just calculated.
To do this correctly in this case, we would want to pour about 150 mL of H2O, add the 0.2 g of peptone, and then bring the final volume up to 200 mL by adding more H2O.
In a hobbyist setting, most fungi being cultivated won't really mind the slight change in concentration if the addition is performed incorrectly; however, if someone sincerely wants to make informed decisions and is going to work with antibiotics, they should probably be maintaining consistency, accuracy, and precision by doing things the correct way. If you skimp and cut corners, don't be surprised if the situation turns out well outside the ideal.
-------------------- Mycology is a cool community of people. Try not to add any bad vibes to the neighborhood. Finding mushrooms in the woods and growing them at home brings me a lot of joy.
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Funguy182
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Registered: 09/04/17
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Last seen: 5 years, 11 days
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Re: Gentamicin question [Re: ChemBioWiz]
#25051906 - 03/09/18 12:54 PM (5 years, 11 months ago) |
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Well I do thank you very much for all of your help it's been so long since I've done math like that which I'm pretty sure is algebra basic algebra I have not done the foil method or please excuse your dear Aunt Sally in many of years so I do appreciate you solving X for me I'm going to go ahead and give it a try thanks again
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Funguy182
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Registered: 09/04/17
Posts: 407
Last seen: 5 years, 11 days
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Re: Gentamicin question [Re: Funguy182]
#25051909 - 03/09/18 12:55 PM (5 years, 11 months ago) |
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I bought both vegetable and meat peptone and boy they both smell like shit the meat peptone more so but that stuff stinks to high heaven especially when I put it in boiling water
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